REVIEWS

Pathogenic mechanisms in HBV-

and HCV-associated hepatocellular
carcinoma
Alla Arzumanyan1, Helena M. G. P. V. Reis2 and Mark A. Feitelson1
Abstract | Hepatocellular carcinoma (HCC) is a highly lethal cancer, with increasing
worldwide incidence, that is mainly associated with chronic hepatitis B virus (HBV) and/or
hepatitis C virus (HCV) infections. There are few effective treatments partly because the
cell- and molecular-based mechanisms that contribute to the pathogenesis of this tumour
type are poorly understood. This Review outlines pathogenic mechanisms that seem to be
common to both viruses and which suggest innovative approaches to the prevention and
treatment of HCC.

Hepatitis
The accumulation of
inflammatory cells in the liver.
Fibrosis
The accumulation of
extracellular matrix material in
the liver that eventually forms
septa.
Cirrhosis
Occurs when fibrotic septa
completely surround islands of
hepatocytes.
1
Department of Biology and
Sbarro Health Research
Organization, College of
Science and Technology,
Temple University,
1900 N. 12th Street,
Philadelphia, Pennsylvania
19122, USA.
2
MIT Portugal Program,
Av. Rovisco Pais, 1049–001
Lisboa, Portugal.
Correspondence to M.A.F. 
e‑mail: feitelso@temple.edu
doi:10.1038/nrc3449
Hepatitis B virus (HBV) (FIG. 1) and hepatitis C virus
(HCV) (FIG. 2) are unrelated viruses that target the liver
and that replicate in hepatocytes. Around 2 billion people
are infected with HBV, and more than 350 million have
become chronic carriers1,2 who have persistent virus and
subvirus particles in their blood for more than 6 months3.
Most infections occur at birth and more than 90%
become chronic. However, only 5–10% of adults who
acquire infection in adulthood become carriers, but up
to 30% develop progressive chronic liver disease (CLD),
which appears as hepatitis, fibrosis, cirrhosis and finally
hepatocellular carcinoma (HCC)4 (FIG. 3). Disease pro‑
gression can arrest at any stage. The risk of developing
HCC among carriers with CLD ranges from tenfold to
100‑fold greater compared with uninfected people2,5,6
depending on the markers and populations that are eval‑
uated. This is one of the closest relationships between an
environmental agent and a cancer that has so far been
identified. The natural history of HCV is also variable,
with up to 85% of acute infections becoming chronic
(FIG. 3). About 50% of people with chronic infections
(or 170 million worldwide) develop CLD, with 5–20%
progressing to cirrhosis within 5–20 years, and 1–2% of
these patients developing HCC per year 7. About 80%
of HCC is due to HBV and/or HCV infections. There are
more than 250,000 new cases of HCC and an estimated
500,000–600,000 deaths due to this disease annually1,8.
The incidence of HCC is increasing, so these infections
remain an important public health burden. In the United
States, Europe, Egypt and Japan, more than 60% of HCC
is associated with HCV, about 20% is related to HBV
and chronic alcoholism contributes to the remainder.
In Africa and Asia, where HBV is endemic, 60% of HCC
is associated with HBV, 20% is related to HCV and the
remainder is distributed among other risk factors (for
example, alcohol and aflatoxin). Most treatments are
inadequate and mortality is high. Patients with small,
single tumours are treated by surgical resection or liver
transplantation, but relapse is common9. Localized and
systemic radiation and chemotherapy have been used,
but with limited success, as they do not target tumour-
associated pathways and because growth-stimulatory
pathways may override the effects of these cytotoxic
therapies. The multi-kinase inhibitor sorafenib has
extended the survival of patients with HCC by about
3 months10, but there is a long way to go before thera‑
pies that target the pathways that drive HCC are clini‑
cally available. Thus, it is timely to review the pathogenic
mechanisms of HBV- and HCV-associated HCC, which
have now been delineated far enough to begin discuss‑
ing common pathways that may be tumour specific.
The goal is to encourage the development of early bio‑
markers and specific intervention strategies that aim to
reduce the risk of CLD and HCC in patients who are
chronically infected.
There are still many challenges ahead. With the
development of rapid and sensitive screening tests, these
viruses have been eliminated as agents of post-transfusion
hepatitis. However, they are still widely transmitted by
intravenous drug abuse, and to a lesser extent are also
sexually transmitted11,12. HBV has a highly efficacious
vaccine, but the implementation of vaccination pro‑
grammes is not uniform, and in endemic countries the
carrier frequency may still exceed 10% of the population.

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REVIEWS
At a glance
• Hepatitis B virus (HBV) and hepatitis C virus (HCV) are major aetiological agents of
chronic liver disease and hepatocellular carcinoma (HCC). HCC is the fifth most
prevalent tumour type and the third leading cause of cancer-related deaths
worldwide, which is why it is so important to find early diagnostic markers and
therapeutic targets, particularly when these markers and targets are common to both
chronic infections.
• Alterations in multiple signalling pathways and patterns of host gene expression have
been documented following HBV and HCV infections, but their relative importance which mediate hepatocellular damage, but they are not
to the pathogenesis of HCC has not been clearly defined. This has limited the ability to
design appropriate therapeutic intervention strategies, and to determine the best
time during chronic infection for their application.
• The pathogenesis of HBV and HCV infections is generally immune mediated, although
these viruses have evolved multiple mechanisms to escape immune elimination and
to continue replicating in an infected host for many years.
• Chronic infection with either virus can result in inflammation and oxidative stress. ber of patients who are infected with HBV23 or HCV24,
A prolonged fibrotic response, resulting in cirrhosis, is also common in both infections,
which is accompanied by the appearance of localized hypoxia, rearrangement of
tissue architecture (epithelial–mesenchymal transition) and angiogenesis.
• Altered host gene expression in chronic liver disease may be mediated by
epigenetic changes, by inhibition of DNA repair and/or by differential expression
of microRNAs. These alterations include constitutive upregulated expression of
factors involved in ‘stemness’, suggesting that both viruses may contribute to HCC by
promoting stemness.
• Early biomarkers and tumour-specific treatments for HCC are mostly lacking, although
several signalling cascades that are activated in the liver before tumour appearance
suggest that oncogene addiction may be important to the pathogenesis of HCC.
• Understanding common mechanisms of HBV and HCV pathogenesis will help to focus
efforts on therapeutic targets that may be most useful in the development of new
treatment approaches.
Although issues of cost, non-compliance, vaccine non-
responders and vaccine-escape mutants have limited
the effectiveness of the vaccine, universal vaccination
is an important goal. In Taiwan, HBV vaccination has
Nucleoside analogues
decreased the incidence of new infections and HCC13.
Derivatives of standard
nucleosides that are There is no vaccine for HCV partly because infections
incorporated into DNA or RNA consist of a genetically heterogeneous ‘swarm’ of virus
during virus replication and particles, some of which escape neutralization14 (FIG. 3).
that bring about chain Although potent antiviral nucleoside analogues against
termination, thereby effectively
inhibiting virus replication.
the HBV and HCV polymerase, as well as protease
inhibitors against HCV, have been developed, drug
HBV-transgenic mice resistance is still a problem. Medium-term nucleoside
Mice generated by introducing analogue treatment of chronic HBV has considerably
a larger than full-length
reduced the incidence of HCC15. For HCV, none of the
molecular clone of hepatitis B
virus (HBV) DNA into fertilized new drugs has been in use long enough to see an effect,
ova, which are then transferred and in fact one of the most promising nucleoside ana‑
into the uterus of logues against HCV that was developed by Bristol-Myers
pseudopregnant female mice. Squibb (BMS‑986094) has been withdrawn from clinical
The offspring stably replicate
HBV from virus RNA made by
trials owing to toxicity 16.
one or more virus DNA
templates that are integrated Pathogenesis
into the host DNA. These mice HBV- and HCV-induced HCC develops in an environ‑
resemble human chronic
ment of inflammation and regeneration that results from
carriers in that they
persistently replicate virus, but chronic liver damage, suggesting that the pathogenesis of
do not develop liver disease or HCC is immune mediated17 (FIG. 3). These viruses persis‑
hepatocellular carcinoma. tently replicate in cell culture without overt cellular dam‑
age or death, implying that they are noncytopathic18,19,
Episome
A virus nucleoprotein complex
although HCV may also have cytopathic properties17.
that replicates independently Persistent virus replication is a risk factor for HCC20
of host chromatin. because inflammation often results in prolonged CLD
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© 2013 Macmillan Publishers Limited. All rights reserved
without virus clearance. In this context, HBV and HCV
generate proteins that blunt the development of immu‑
nity that should resolve chronic infections (see below).
Although virus-specific lymphocytes are readily detect‑
able in inflammatory lesions in the liver, they are often
not sufficient to clear virus infection21. Many virus non‑
specific natural killer (NK) cells, NKT cells and polymor‑
phonuclear leukocytes are also found in these lesions,
effective in virus clearance21. Imbalances in cytokine pro‑
duction may also hinder virus elimination. In chronic
HBV, there seems to be a deficiency in interferon (IFN)
production, and IFN therapy in HBV-transgenic mice has
shown strong anti-viral effects22. However, IFN reduces
virus titres and disease progression in only a small num‑
suggesting that success will require more than simple
cytokine replacement.
HBV- and HCV-encoded proteins alter host gene
expression and cellular phenotypes that are recog‑
nized as hallmarks of cancer. These changes promote
growth factor-independent proliferation, resistance
to growth inhibition, tissue invasion and metastasis,
angiogenesis, reprogramming of energy metabolism, and
resistance to apoptosis in the face of persistent immune
attack and during therapeutic intervention25. Chronic
inflammation also promotes genetic instability in tumour
cells. The contribution of HBV to HCC involves the
expression of hepatitis B x (HBx) and possibly carboxy-
terminally truncated pre-S or S polypeptides (FIGS 1,4,5);
for HCV, the core protein, and non-structural (NS)
proteins NS3 and NS5A contribute to oncogenic trans‑
formation (FIGS 2,4,5). Changes in host gene expression
that promote tumorigenesis also seem to support virus
replication and/or protect virus-infected hepatocytes
from immune-mediated damage or destruction. Thus,
tumour-promoting changes seem to be coincidental, as
the development of HCC does not seem to benefit these
viruses and most often kills the host.
Virus persistence refers to the manner in which HBV
and HCV exist in infected cells over a long period, and
it is important to the pathogenesis of HCC. For HBV,
virus DNA exists as an episome in the form of a mini-
chromosome in the nuclei of cells26. Regeneration of
infected hepatocytes during CLD often results in the
integration of HBx and sometimes truncated S and
pre-S sequences into many sites in the host DNA. The
corresponding HBx and pre-S or S polypeptides have
transforming properties. Although integration into host
DNA may contribute to HCC through cis-acting mecha-
nisms, the large number of integration events at different
sites in host DNA does not support cis activation as a
major mechanism in HCC. Instead, the products trans‑
lated from the integrated templates more commonly
affect host gene expression by trans-acting mechanisms.
Intracellular levels of HBx often increase after each cycle
of hepatocellular regeneration (FIG. 4). By contrast, HCV
persistence does not include integration, and maintains
itself as an endoplasmic reticulum (ER)-associated epi‑
some. In infected hepatocytes, these viruses encode pro‑
teins that promote survival and growth, which favour
 REVIEWS

Cis-acting mechanisms the expansion of virus-infected cells following each bout reciprocal interference: when HBV markers are present,
Occur when viral DNA of hepatitis. Thus, progressive CLD seems to be asso‑ HCV markers are mostly or completely absent from
integration results in the ciated with an increase in the proportion of virus- the liver and blood. In this case, the suppressed agent
altered expression of a gene positive cells in the liver that acquire the characteristics assumes an occult infection3,28. In occult HBV infections,
adjacent to or close to the site
of integration.
of tumour cells27. the HCV core protein binds to HBx, blocking its trans‑
Persistent co‑infections in a single patient seem to activation function, and also binds to the HBV poly‑
Trans-acting mechanisms accelerate the pathogenesis of HCC through common merase and pre-genomic RNA, interfering with HBV
Occur when a virus encodes necro-inflammatory pathways or by direct oncogenic replication29. HBx in occult HBV infections stimulates
one or more proteins that
activity 28. Both exacerbate oxidative stress and the pro‑ HCV replication30. Some 30–80% of occult HBV infec‑
influence the expression of
host genes at many
duction of cytotoxic cytokines. In addition, virus muta‑ tions have integrated HBV DNA31 that expresses HBx,
chromosomal sites. tions may affect the robustness of virus replication and such patients have a lower but still increased risk of
and virus–virus interactions. These viruses also show HCC28. Among carriers with HCC, up to 23% are also
anti-HCV-positive, suggesting frequent co‑infection,
especially in countries where both viruses are endemic32.
pre-S2: 55 aa
pre-S1: 128 aa Altered innate and adaptive immunity
HBV and HCV block or alter immune responses, but
– Strand
the outcome of acute infection is influenced by the virus
inoculum, the number of hepatocytes infected and the
+ Strand S: 226 aa kinetics of the induction of innate responses. Toll-like
3,182-1 receptor (TLR) signalling, which triggers innate immunity,
inhibits virus replication in HBV-transgenic mice33.
2,800 400 However, HBV alters TLR signalling 34, resulting in
liver damage that is not effective in virus clearance.
In chimpanzees, acute HBV infection fails to trigger
innate immunity in the first weeks of infection17 (FIG. 3).
A major signalling molecule in innate immunity is nuclear
2,400
factor-κB (NF-κB), which induces pro-inflammatory
800
and hepatoprotective gene expression35. HBx activation
of NF-κB36 may suppress innate immunity by switching
NF-κB signalling to hepatoprotection, thus promoting
resistance to immune-mediated damage. Intrahepatic
NK cell function is also skewed towards cytolytic activ‑
2,000 1,200
ity without IFNγ production, suggesting that hepatocel‑
lular killing occurs without virus clearance37. Dendritic
C: 183 aa DR1 1,600 cells (DCs), which link innate immunity with adaptive
P: 832 aa
DR2 immunity, and which may be infected with HBV, are also
pre-C defective in chronic HBV infection, resulting in poor
adaptive immunity 38. CD4+CD25+FOX3P+ regulatory T
(TReg) cells help to maintain a tolerogenic environment in
the liver. These TReg cells are induced by HBx-stimulated
production of transforming growth factor‑β1 (TGFβ1)39.
In adaptive immunity, there are also defects in HBV-
X: 154 aa
specific CD8+ T cells40, which are characteristic of T cell
Figure 1 | The HBV genome and encoded genes.  Hepatitis B virus (HBV) is a small exhaustion.
DNA virus with a 3.2 kb genome. Virus particles have a partially Nature
double-stranded genome
Reviews | Cancer By contrast, HCV-infected chimpanzees show early
(indicated by a dashed line) with a cohesive overlap that spans the 5ʹ regions of each
strand and that is flanked by direct repeat sequences (DR1 and DR2). The virus encodes changes that are indicative of innate immunity 17. HCV
proteins from four genes on the minus strand of HBV DNA181. The gene S encodes the activates TLR3, which senses double-stranded RNA
major hepatitis B surface antigen (HBsAg) protein and its glycosylated partner, which are (dsRNA) in virus-replication intermediates, and retin‑
transmembrane proteins in the virus envelope. In‑frame sequences upstream from the S oic acid-inducible gene 1 (RIG1), which is an RNA heli‑
gene encode the pre-S domains, which are translated with the S sequences to make the case that recognizes the polyuridine motif of the HCV
pre-S and S polypeptides (middle and large proteins) that contain the virus receptor for 3′ untranslated region (UTR)41. Subsequent signalling
infection of hepatocytes. Gene C encodes the hepatitis B core antigen (HBcAg) which via NF-κB results in IFNβ production, which triggers
forms the nucleocapsid of the virus. The P region encodes the virus reverse transcriptase Janus kinase (JAK)–signal transducer and activator of
that also has DNA-dependent DNA polymerase activity and RNase H activity required for transcription (STAT) signalling in neighbouring cells,
virus replication. Although HBV is a DNA virus, it replicates through a pre-genomic RNA
and in the activation of IFN-target genes that promote
intermediate. Finally, the X gene encodes the small regulatory protein of the virus, the
hepatitis B x (HBx) antigen. HBx is a transactivating protein that stimulates virus gene the degradation of viral and cellular RNAs42. However,
expression and replication, protects virus-infected cells against immune-mediated HCV NS3–4A block TLR3 and RIG1 (REF. 43). In addi‑
destruction and contributes to the development of hepatocellular carcinoma. aa, amino tion, HCV core inhibits JAK–STAT44, and HCV NS5A
acid. Image is modified, with permission, from REF. 181 © (1985) Macmillan Publishers and E2 inhibit IFN signalling 45,46. Thus, innate immu‑
Ltd. All rights reserved. nity is triggered, but it is blocked by HCV (FIG. 3). With

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REVIEWS

a 5′ UTR 3′ UTR
III
IRES
II
Variable region

ORF
5′ Poly(U/UC) tract
I
IV

Translation and processing

b
Core E1 E2 p7 NS2 NS3 NS4A NS4B NS5A NS5B
1 192 384 747 810 1,027 1,658 1,712 1,973 2,419 3,010

Cleavage by cellular signal Autoproteolytic

Cleavage by HCV NS3
peptidase or peptidases cleavage

Figure 2 | The HCV genome and encoded proteins.  a | Hepatitis C virus (HCV) is a positive, single-stranded RNA virus
Nature Reviews | Cancer
that encodes a large polyprotein of about 3,000 amino acids from a single open reading frame (ORF). The ORF is flanked by
two untranslated regions (UTRs), which contain signals for viral protein and RNA synthesis, and for the coordination of
both processes. Translation is initiated through an internal ribosomal entry site (IRES) in the 5ʹ UTR. b | In polyprotein
maturation, the core–E1, E1–E2, E2–p7 and p7–non-structural 2 (NS2) junctions are cleaved by one or more cellular signal
peptidases to yield the structural proteins of the virus. The structural proteins of HCV consist of the core protein and the
two envelope glycoproteins (E1 and E2). p7 is an ion channel protein that promotes virus assembly. The NS2–NS3 proteins
undergo autocatalytic cleavage, which releases the NS3 serine protease. NS3 cleaves the remainder of the non-structural
polypeptides, which then assemble into membrane-associated replication complexes. NS5B (an RNA-dependent RNA
polymerase) then replicates the viral RNA. NS4B and NS5A have less well-defined roles in virus replication. NS3 also has
helicase and NTPase activities that are important for virus replication. The resulting proteins fold into a replication
complex and the mature virus buds through the membrane of the endoplasmic reticulum. The core protein, NS3 and NS5A
may also contribute to hepatocarcinogenesis. Figure is modified, with permission, from REF. 182 © (2009) American
Society for Clinical Investigation.

Oxidative stress
An increase in the intracellular
levels of reactive oxygen
species (ROS), which are
associated with most
pathological states, particularly
those involving inflammation.
Virus inoculum
The amount of virus that an
individual is exposed to during
acute infection.
Innate immunity
Comprises responses that
develop rapidly, but that are
antigen nonspecific. Often
mediated by molecules binding
to toll-like receptors.
Adaptive immunity
Comprises responses that are
primed by innate immunity
and that are strong and antigen
specific.
Tolerogenic environment
An environment in which
immune responses are not
readily triggered against
antigens.
regard to adaptive immunity, chronic HCV infection
is characterized by virus-specific CD8+ T cells in the
liver — but they do not consistently correlate with dis‑
ease severity 47 — and by weak CD4+ T cell proliferative
responses48. Hopefully, pharmacological suppression
of HCV NS3–4A and NS5B may result in the restoration of
strong immune responses that aim to eliminate the virus.
Both HBV and HCV encode proteins that have doc‑
umented pro-apoptotic and anti-apoptotic properties,
depending on the experimental system used. Although
controversial, it seems that when these viruses infect
quiescent hepatocytes and promote inappropriate cell
growth, then the cellular response is to trigger growth
arrest or apoptosis; however, when these viruses infect
liver progenitor cells, or liver cells with defective nega‑
tive growth regulatory circuitry, growth arrest and apo‑
ptosis do not occur 49,50. Whether apoptosis occurs may
also depend on the intracellular concentration of the
virus oncoproteins. Among infected hepatocytes with
low levels of virus oncoproteins, cell cycle arrest and
apoptosis predominate, but among cells with progres‑
sively higher intracellular levels of virus oncoproteins,
increasingly strong survival and proliferative signals
override negative growth regulation, resulting in resist‑
ance to apoptosis. Resistance to apoptosis not only pro‑
motes virus persistence, but it is also characteristic of
tumour cells (FIG 4). For HBV, sustained high levels of
HBx block tumour necrosis factor-α (TNFα)- and FAS-
mediated apoptosis by activation of NF‑κB51, suggesting
that infected hepatocytes survive immune-mediated
damage, but that uninfected hepatocytes undergo
extensive apoptosis in CLD. This may explain why
HBx expression correlates with the severity of CLD27.
The intra­cellular concentration of HBx increases with
additional integration events following each bout of
hepatitis and regeneration, resulting in outgrowth of
HBx-positive hepatocytes. For HCV, the overexpres‑
sion of core and NS5A blocks apoptosis by activation
of AKT and NF‑κB, respectively 52,53. In chronic HCV,
FAS is upregulated in hepatocytes54, which may promote
the survival of virus-infected cells by triggering apopto‑
sis in inflammatory T cells. These events protect cells
from immune-mediated elimination, are pro-oncogenic
and may help to explain how these viruses persist in the
liver despite repeated bouts of hepatitis that should have
eliminated the virus.
Oxidative stress
HBx and the HCV core protein are associated with mito‑
chondria55,56 where they trigger oxidative stress that may
result in apoptosis57,58 (FIG. 4). Proteomic and metabolite
profiles from HBV-transgenic mice have shown evi‑
dence of oxidative stress59. In these mice, oxidative stress
triggered an antioxidative response, exemplified by the
activation of forkhead box O4 (FOXO4), which confers
resistance to oxidative cell death60. In HBV replication,
HBx promotes cytosolic calcium signalling, resulting in
the accumulation of Ca2+ in mitochondria and increased
levels of reactive oxygen species (ROS)61, as well as the
activation of the PYK2 and SRC kinases, which both

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Acute, resolving infection the ER, followed by uptake into mitochondria, result‑
ing in decreased mitochondrial permeability transition,
and the generation of ROS65. In both cases, activation
Rapid, strong, multi-specific
immune responses of ROS-sensitive transcription factors stimulates virus
25% 15% replication and cell growth, and contributes to tumour
development (FIG. 4).
HBV HCV When ER stress occurs, protein-folding capacity is
10% 85% compromised and the unfolded protein response (UPR)
Fails to trigger Blocks innate
innate immunity immunity is activated to correct the protein defect and to restore ER
function. Both HBV (HBs and HBx) and HCV (NS3–4A)
alter Ca2+ signalling and increase ROS levels, which trigger
Slow, weak adaptive immunity
HBx pre-S and S to few virus antigens resulting NS3 NS5A Core ER stress and the UPR66,67 (FIG. 4). However, when ER stress
in virus persistence becomes chronic during virus infection, UPR signalling
cannot be sustained, and autophagy is activated to help to
70% 50%
degrade the unfolded proteins and to maintain cell viabil‑
ity. Autophagy is triggered by both viruses67,68 in order to
Asymptomatic Asymptomatic help restore ER integrity, which is important for viral rep‑
carriers 30% 50% chronic infections
lication67,69. Autophagy also promotes cell survival70 and
virus persistence, which are risk factors for HCC71 (FIG. 4).
May resolve Hepatitis
Hypoxia and angiogenesis
May regress or
In chronically infected livers, the development of cir‑
Fibrosis
become inactive rhotic nodules is accompanied by a progressive decrease
of vasculature. This results in localized regions of reduced
Cirrhosis oxygen tension, and the development of a hypoxic
environment (FIG. 4). The cellular response to hypoxia
Promotes hallmark changes of cancer involves upregulated expression of HIF1α, which pro‑
motes angiogenesis by transcriptionally upregulating
vascular endothelial growth factor (VEGF), elevates
HCC
pro-inflammatory prostaglandin levels by stimulating
Figure 3 | Immunity in the natural history of HBV and HCV infections.  Immunity has cyclooxygenase 2 (COX2) and enhances cell migration
an important role in the outcome of acute infections. Rapid, strong Nature Reviews | Cancer
and multi-specific by activation of matrix metallo­proteinases (MMPs)72.
responses against many hepatitis B virus (HBV) and hepatitis C virus (HCV) proteins often In HBV infection, HBx binds to and stabilizes HIF1α
result in an acute infection that is cleared by the host. The failure of HBV to trigger innate
and stimulates HIF1α transcription73,74, thus promot‑
immunity results in suboptimal priming of adaptive immune responses that are crucial to
ing angiogenesis and cell ‘stemness’ (REF. 75) (see below).
virus clearance, and this promotes the onset of chronic infections. Before the HBV vaccine,
this was most likely to occur in infants and young children with immature immune HBx also promotes angiogenesis by stimulating the ERK
systems. Approximately 65% of acute HBV infections of adults are subclinical; about 1% of MAPK, which upregulates the pro-angiogenic growth
acute adult infections result in fulminant hepatitis, which is rapid, highly aggressive and factor angiopoietin 2 (ANG2)76 (FIG. 4). HCV core, E1,
may be fatal (not shown). For HCV, innate immunity is triggered on acute infection, but is NS3 and NS5A also upregulate HIF1α under hypoxic
blocked by the virus, again resulting in poor adaptive immunity. In addition, acute HCV conditions, resulting in increased VEGF, COX2, ANG2
infections consist of a ‘swarm’ or ‘quasi-species’ in which different virus particles consist of and several MMPs72,76,77 (FIG. 4). Hypoxia also aggravates
slightly different sequences in their neutralizing determinants. The most prevalent of cell damage and inflammation, inhibits liver regenera‑
these quasi-species trigger neutralizing antibodies, but by the time this happens, minor tion and is a major stimulus in the pathogenesis of HCC.
quasi-species become dominant and new quasi-species arise owing to the lack of
The importance of angiogenesis has been highlighted by
proofreading by the virus-encoded polymerase during virus replication. The result is that
the use of the multikinase inhibitor sorafenib10, which
the virus is often one step ahead of the ability of the host to develop neutralizing
antibodies, and this often contributes to the appearance of chronic infections. HBx, targets the VEGF receptor (VEGFR) and other signal‑
hepatitis B x; HCC, hepatocellular carcinoma; NS, non-structural. ling pathways. However, it has yielded only transitory
improvements followed by the appearance of resistance.
HIF is also stabilized by increased insulin-like growth
promote HBV replication and participate in early steps factor receptor 1 (IGFR1), epidermal growth factor recep‑
of HCC62 (FIG. 4). HBx in mitochondria binds to the tor (EGFR) and PI3K–AKT signalling. Both EGFR and
HIF1 voltage-dependent anion-selective channel protein 3 PI3K–AKT signalling are activated by HBx and HCV
A transcription factor (VDAC3), leading to membrane depolarization and to (FIG. 4). Increased EGFR signalling seems to mediate the
comprised of an α-subunit and
the production of additional ROS56. transition from cirrhosis to HCC in rats78. EGFR signals
a β-subunit that triggers
changes in gene expression HCV core protein-transgenic mice that developed through heterodimerization with ERBB2. ERBB2 is upreg‑
that promote cell survival HCC had impaired function of the mitochondrial res‑ ulated and co‑stains with HBx in the livers of patients with
under hypoxic conditions. piratory chain, increased ROS levels and decreased HCC, suggesting ERBB2 activation79 (FIG. 4). Increased
function of several antioxidants63. Activation of hypoxia- ERBB2 levels are also present in the livers of chronic
Autophagy
An adaptive response that
inducible factor 1 (HIF1) resulted in the upregulation of HCV-infected and tumour-bearing patients80. These
promotes cell survival under target genes, including those for glycolytic enzymes64. pathways of HIF activation underscore the importance
conditions of stress. In the HCV-infected cell, there is a release of Ca2+ from of hypoxia in the pathogenesis of HCC (FIG. 4).

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Senescence
Occurs when a particular cell
type has undergone a
predetermined number of cell
divisions. These cells remain
metabolically active but no
longer divide.
Oncogenes, tumour suppressors and senescence
Altered expression of senescence-related genes is often
associated with the activation of RAS signalling 81,
which is activated by HBx 82. HBx is also capable of
overcoming RAS-induced senescence in immortal‑
ized cells83. Activation of RAS in pre-malignant tissues
showed that senescence exists before tumour appear‑
ance, suggesting that it may be valuable in the diagnosis
and prognosis of early cancer 81. Oncogene-induced
senescence has been linked to two signalling pathways
that are often disrupted in cancer: AKT–ARF–p53–p21
(also known as WAF1) and RAS–MEK–ERK–INK4A
(also known as p16)–RB, both of which operate in
HCC84 and are activated by HBx 85,86. Thus, overcom‑
ing senescence seems to be a major step in early tumour
development.

a
Low HBx Increased HBV High HBx
levels in liver DNA integration levels in liver

Regeneration Changes in liver architecture

Inflammation Hepatitis Fibrosis Cirrhosis HCC Metastasis

b TGFβ
TGFβR IGFR1 EGFR ERBB2 E-cadherin

RAS–ERK
β-catenin

PI3K–AKT HBx HCV EMT

proteins

Cirrhosis
• RAS–ERK
SMADs to HCC SRC Increased HIF,
• PI3K–AKT Resistance
transition hypoxia and
to apoptosis
angiogenesis

HCV HBx
proteins Stemness VEGF, COX2,
Mitochondrial (NANOG, KLF4, ANG2, MMPs
stress OCT4 and MYC) and NOTCH

HCV
proteins Mitochondria MYC, MDR1
ROS and TERT
HBx
ER
stress
HDAC HBx
Autophagy UPR SMADs
A2M
NF-κB NF-κB
Increased cell FN
SNAIL CDH1
survival and HCC risk
persistent virus factors EMT
replication β-catenin EPCAM
TGFB1
ER
Nucleus

TGFβ

Figure 4 | Signalling pathways targeted by HBV and HCV in CLD pathogenesis. a | Major steps in the
Nature pathogenesis
Reviews | Cancer
of hepatitis B virus (HBV) and hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC) are shown. In both virus
infections, immune responses to persistent virus replication promote the development of hepatitis, which consists of
hepatocellular destruction followed by regeneration and/or fibrosis. During regeneration, the hepatitis B x (HBx) and
pre-S and S genes increasingly integrate into host DNA, resulting in increased levels of intracellular HBx. b | The activities
and expression of selected signalling pathways and target genes are altered by HBV- and HCV-encoded proteins. Note
that both viruses promote the growth and survival of infected cells during chronic liver disease (CLD), and activate several
overlapping signalling pathways (for example, RAS, PI3K, epidermal growth factor receptor (EGFR) and insulin-like growth
factor receptor 1 (IGFR1)) that contribute to the pathogenesis of HCC. A2M, α2‑macroglobulin; ANG2, angiopoietin 2;
CDH1, E-cadherin; COX2, cyclooxygenase 2; EPCAM, epithelial cell adhesion molecule; EMT, epithelial–mesenchymal
transition; ER, endoplasmic reticulum; FN, fibronectin; HDAC, histone deacetylase; HIF, hypoxia-inducible factor; TERT,
human telomerase reverse transcriptase; MDR1, multi-drug resistance 1; MMP, matrix metalloproteinase; NF-κB, nuclear
factor-κB; ROS, reactive oxygen species; TGFβ, transforming growth factor-β; UPR, unfolded protein response; VEGF,
vascular endothelial growth factor.

128 | FEBRUARY 2013 | VOLUME 13 www.nature.com/reviews/cancer

© 2013 Macmillan Publishers Limited. All rights reserved

 REVIEWS

Inflammation, oxidative and oncogenic stress in These viruses overcome senescence by inhibiting the
chronic HBV and HCV infections accelerate cellu‑ expression and function of tumour suppressors (FIG. 5).
lar senescence. Senescence limits the proliferation of Both viruses upregulate DNA methyltransferases
damaged cells and reduces the risk of malignancy 87 (DNMTs)89,90, which decrease the expression of genes
by triggering the expression of tumour suppressors88. involved in DNA repair 91. HBx, as well as the HCV
core, NS3 and NS5A proteins, compromise several
a p53 functions53,92, including nucleotide excision repair
HBx β-catenin miR-181 TIMP3 and transcription-coupled repair 93. HBx also promotes
the methylation of the ankyrin-repeat-containing,
• MMP2 SH3‑domain-containing and proline-rich-region-
Tumorigenicity
• MMP9
containing proteins (ASPP1 and ASPP2) that activate
EPCAM Stemness p53 (REF. 94). HCV also blocks DNA repair through the
ataxia telangiectasia mutated (ATM) DNA repair path‑
miR-148a PTEN PI3K–AKT–mTOR Loss of tumour way 95. In addition, HCV core protein and HBx suppress
suppressors, DNA the cyclin-dependent kinase (CDK) inhibitors INK4A
repair and genome and p21 via promoter methylation, resulting in the
miR-373 CDH1 EMT integrity
inactivation of the RB tumour suppressor 96,97 (FIG. 5).
p53 Transcription- A microRNA (miRNA), miR‑221, which is upregulated
ERCC3
coupled repair in HBV- and HCV-related HCC, blocks the expression
of the CDK inhibitor p27 and promotes tumour growth
HCV miR-141 DLC1 and progression by activation of the PI3K–AKT–mTOR
proteins
pathway 98. Thus, abrogating the pathways that are
populated by tumour suppressors and CDK inhibitors
b HBx TFIIH p53 compromises the ability of cells to mediate DNA repair,
DNA excision repair thereby promoting genetic instability 99.
HCV
proteins DNMT ATM The ability of HBV and HCV to alter the expres‑
sion of selected miRNAs affects virus persistence,
miR-602
as well as oncogene and tumour suppressor path‑
RASSF1A ways in HCC (FIG. 5). For example, miR‑122, which
Loss of tumour
suppressors, DNA promotes hepatocellular differentiation and tumour
repair and genome suppression, blocks cyclin G1 expression and inhibits
miR-152 DNMT1 INK4A RB integrity HBV replication by a miR‑122–cyclin G1–p53–HBV
CDKN1A
enhancer regulatory pathway 100. HBV increases miR‑122
expression, which targets HBx sequences101, thereby
ASPP1 and ASPP2 p53 promoting virus persistence. For HCV, miR‑122
stimulates virus replication by stabilizing the virus
CDH1 EMT RNA in infected liver 102, and is suppressed in HCC100,
which may explain why HCV replication is generally
GSTP1 ROS AP1 miR-21
lower in HCC compared with adjacent liver. However,
miRNA arrays using virus-infected compared with
uninfected liver showed that pathways that are related
Inflammation
miR-122 Virus persistence and metastasis
to cell death, DNA damage, recombination and sig‑
nal transduction were activated in HBV-infected liver,
Figure 5 | Examples of epigenetic alterations in host gene expression that are and those related to immune response, antigen pres‑
relevant to the pathogenesis of HBV- and HCV-associated HCC. Part a shows a few entation, proteasome, cell cycle and lipid metabolism
of the pathways that distinguish the mechanisms through which hepatitis
Nature B virus
Reviews (HBV)
| Cancer
were activated in HCV-infected liver 103. Although
and hepatitis C virus (HCV) contribute to hepatocarcinogenesis, and part b illustrates
some of the shared pathways. Hepatitis B x (HBx)-upregulated expression of the these data suggest differences in the mechanisms
microRNA (miRNA) miR‑181 promotes tumorigenicity, and upregulated expression of of HCC used by these viruses, many miRNAs target
miR‑148a and miR‑602 blocks the function of crucial tumour suppressor proteins. HBV overlapping signalling pathways and genes, suggesting
and HCV upregulation of miR‑21 in an environment of oxidative stress promotes that further analysis may reveal common pathways
inflammation in the liver and metastases during tumour progression. Both viruses also of oncogene activation and inactivation of tumour
affect p53 and RB function, resulting in the loss of genome integrity and uncontrolled suppression.
cell cycle progression, respectively. Thus, inactivation of tumour suppressors and the
activation of oncogenic pathways, some of which are regulated via virus alterations in EMT and fibrogenesis
miRNA expression profiles, seem to be important to hepatocarcinogenesis. AP1, Epithelial–mesenchymal transition (EMT) is important
activating protein 1; ATM, ataxia-telangiectasia mutated; CDH1, E-cadherin; CDKN1A,
in the pathogenesis of CLD as it promotes fibrogenesis,
cyclin-dependent kinase inhibitor 1 (encodes p21); DLC1, deleted in liver cancer 1;
DNMT1, DNA methyltransferase 1; EPCAM, epithelial cell adhesion molecule; ERCC3, tumour progression and metastasis. In fibrogenesis, type 2
excision repair cross-complementing rodent repair deficiency, complementation EMT occurs in response to continuing inflammation,
group 3; GSTP1, glutathione S‑transferase P1; MMP, matrix metalloproteinase; ROS, ultimately leading to organ destruction. De novo expres‑
reactive oxygen species; TFIIH, transcription factor II H; TIMP, tissue inhibitor of sion of mesenchymal proteins104 — such as fibroblast-
metalloproteinase 3. specific protein 1 (FSP1; also known as S100A4),

NATURE REVIEWS | CANCER VOLUME 13 | FEBRUARY 2013 | 129

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REVIEWS
Epithelial–mesenchymal
transition
(EMT). A transient and
reversible switch from a
polarized, epithelial phenotype
to a fibroblastoid or
mesenchymal phenotype. EMT
is divided into three categories:
type 1 occurs in development
(associated with embryo
implantation, formation and
organ development); type 2
occurs in fibrosis (associated
with tissue damage and
inflammation, and generates
repair-associated
mesenchymal cells and
fibroblasts); and type 3 occurs
in cancer and metastasis.
Fibrogenesis
Involves the synthesis and
deposition of extracellular
matrix proteins, and is a form
of tissue repair that follows a
bout of hepatitis, in which
hepatocytes are injured and
destroyed.
Cancer stem cells
(CSCs). A subset of tumour
cells that share properties with
normal tissue stem cells,
including self-renewal (by
symmetric and asymmetric
division) and the capacity to
differentiate.
Epithelial cell adhesion
molecule
(EpCAM). A cell adhesion
molecule found on hepatic
stem cells and cancer stem
cells; when activated by
proteolysis, an intracellular
domain reaches the nucleus,
where it triggers cell cycle
progression and mitosis by
upregulating cyclin E and MYC
expression.
Telomerase reverse
transcriptase
(TERT). Helps to prevent the
erosion of chromosomal ends
(telomeres) after each cycle of
cell division. Constitutive
overexpression of telomerase
(of which TERT is a component)
is characteristic of many
tumour types.
130 | FEBRUARY 2013 | VOLUME 13 www.nature.com/reviews/cancer
collagen I and collagen III, and α‑smooth muscle
(α‑SMA) — and upregulated expression of base‑
ment membrane-degrading MMP2 and MMP9 by
hepatic stellate cells (HSCs)105 that transdifferenti‑
ate into myofibroblasts 106 (and possibly hepatocytes
having undergone EMT)107 retain a permanent mes‑
enchymal state that contributes to fibrosis108. TGFβ,
which promotes EMT by regulating transcription
factors that suppress epithelial markers (for example,
components of cell junctions) and which enhances
mesenchymal features, is central to the generation
of myofibroblasts. Once activated, myofibroblasts
secrete TGFβ and sustain their own activation by
an autocrine mechanism. TGFβ1 promotes the tran‑
scription of genes encoding collagen I and collagen
III 109, as well as SNAIL 110, which transcriptionally
suppresses E‑cadherin 111 (FIG.  4) . HBx upregulates
TGFβ1 (REF. 39) by SMAD-dependent (via stabili‑
zation of the SMAD4 complex) 112 and non-SMAD-
dependent pathways (via activation of RAS–ERK
and PI3K–AKT)113. HCV core protein activation of
TGFβ34 also promotes EMT in hepatocytes and HCC
cells. HBx and HCV core also seem to convert TGFβ1
signalling from negative to positive growth regulation
and shift TGFβ responses from tumour suppression
to EMT112,114.
Liver fibrogenesis (type 2 EMT) and HCC metasta‑
sis (type 3 EMT) are also mediated by miR‑21, which
is upregulated by NF-κB in HBV- and HCV-associated
HCC115. Activated TGFβ and RAS–ERK–AP1 result
in the inhibition of multiple tumour suppressors116.
Importantly, these signalling pathways are also acti‑
vated by HBx and HCV 36,53,80,82,86,112,114. As miR‑21
activation usually occurs during CLD, in which ROS
stimulates NF-κB and AP1, these observations provide
a link between chronic inflammation and early events
in HCC.
SRC signalling, which is activated by both viruses,
is important to EMT 117,118 (FIG. 4). SRC is known to
promote the expression of MMPs by multiple mecha‑
nisms, including activation of the ERK and PI3K
pathways. HBx-stimulated SRC promotes EMT119 by
destabilization of adherens junctions120. This is asso‑
ciated with tyrosine phosphorylation of β‑catenin
(activation) in E‑cadherin complexes 120. In addi‑
tion, E‑cadherin is suppressed by the HBx-mediated
upregulation of SNAIL121, and HCV (NS5A) upregu‑
lation of TWIST2 (REF. 122). E‑cadherin expression
is also suppressed by promoter DNA methylation,
which is mediated by HBx 121 and the HCV core pro‑
tein 123 (FIG. 5). Further, these viruses promote EMT
by activation of RAS, WNT and VEGF signalling,
which all activate SNAIL. HCV core and E2 have
also been implicated in fibrogenesis by triggering
EMT 124,125. Thus, activated SRC signalling by these
viruses contributes to early steps in HCC. During
natural infection, fibrosis may resolve spontaneously
or may resolve as a result of sustained inhibition of
virus replication, but generally, advanced fibrosis and
cirrhosis may become inactive (that is, arrested) but
not resolved.
© 2013 Macmillan Publishers Limited. All rights reserved
Do these viruses promote stemness?
Both viruses promote characteristics of cancer stem
cells (CSCs) (FIG. 4), which have an important role in
the pathogenesis of HCC126–129. Stemness markers (such
as NANOG, OCT4, SOX2 and Krüppel-like factor 4
(KLF4)) are reactivated130,131 and expressed in HCC132,
as well as in many other tumour types 133. HBx pro‑
motes the expression of NANOG, KLF4, OCT4 and
MYC, as well as the stemness-associated markers epi-
thelial cell adhesion molecule (EpCAM) and β‑catenin132.
Although normal adult hepatocytes do not express
EpCAM, HBV-infected hepatocytes do express this
protein, implying that HBx confers stemness properties
to at least some infected cells132. NANOG and MYC,
as well as the stemness-associated markers β‑catenin,
CD‑133 and LIN28, were also upregulated by HCV134.
Stabilization of β‑catenin promotes the transcription
of stemness genes in association with TCF/LEF1,
OCT4 and NANOG135. β‑catenin also transcriptionally
upregulates EpCAM132. EpCAM, in turn, contributes
to stemness through its binding to OCT4, NANOG,
SOX2 and KLF4 promoter regions136. In HCC xeno‑
graft experiments, only EpCAM+ cells efficiently gener‑
ated invasive tumours, suggesting that EpCAM+ cells
display CSC-like properties such as tumour initiation
and progression137. Both HBV and HCV promote the
expression of one or more miR‑181 family members,
which are known to upregulate EpCAM129,138 (FIG. 5).
miR‑181 members are highly expressed in embry‑
onic livers, in HSC, and in patients with α-fetoprotein
(AFP)-positive tumours, suggesting that they help to
maintain stemness138.
Stemness is favoured by hypoxia, which enhances
the activity of OCT4, MYC and NANOG139. The effect
of hypoxia on CSCs is primarily mediated by HIFs.
Although the detailed mechanisms that link hypoxia to
stemness are not known, β‑catenin can enhance the tran‑
scription of HIF1A, thereby promoting cell survival and
adaptation to hypoxia140. HIF upregulates MYC, which
transactivates human telomerase reverse transcriptase
(TERT)141, thus linking two key regulators of stem cell
biology and cancer. Hypoxia also stimulates EMT in
HCC142 and in hepatocytes, suggesting that HIF1α
(which is upregulated by both viruses)73,74,77 (FIG. 4) is
important for EMT143. In other tumour types, EMT pro‑
vides an environment that promotes the appearance of
CSCs144 that may facilitate escape to a new niche. EMT
and stemness share several signalling pathways that are
constitutively activated by these viruses (FIG. 4). In ovarian
cancer, the acquisition of stemness ensures the generation
of the crucial tumour mass required for progression and
metastases145. This may also occur in HCC. The finding
that EMT generates self-renewing stem cells provides
a link between tumour metastasis and CSCs. In other
words, cancer cells that undergo an EMT are capable of
metastasizing through their acquired invasiveness and
then initiate metastases through self-renewal potential.
Detailed understanding of the origin and molecular pro‑
file of liver CSCs, and how normal stem cells in the liver
differ from CSCs, may allow them to be targeted, thus
improving therapeutic outcome.

Biomarkers and therapies
A major difficulty in developing early biomarkers
of HCC comes from an incomplete understanding of
tumour pathogenesis, and the extent of marker variability
among patients. This is why personalized therapy is not
yet a reality for patients with HCC. In one study, a sub‑
set of patients with HCC who had elevated intrahepatic
EpCAM and β‑catenin levels had a poor prognosis relative
to EpCAM-negative patients with HCC146. Independent
work has shown a considerable difference in the survival
of patients with HCC post-diagnosis based on elevated
expression of ERBB2 (REF. 79). Both EpCAM and ERBB2
are functionally connected to β‑catenin, which promotes
stemness at early stages of HCC. If further work provides
validation that any of these markers have prognostic
value, their detection before tumour appearance may also
suggest their usefulness as therapeutic targets.
Global hypomethylation of DNA also alters gene
expression before tumour appearance, which turns on
genes that promote growth, as does selected hyper‑
methylation of tumour suppressor genes, including
IGF-binding protein 3 (IGFBP3), RASSF1A, INK4A,
E‑cadherin and glutathione S‑transferase P1 (GSTP1)147
(FIG. 4). In chronic HBV infection, HBx suppression of
miR‑152 upregulates DNMT1, which methylates the
promoters of most of these tumour suppressors92,147.
DNA methylation signatures may help to classify
patients for treatment purposes, but their clinical
usefulness remains to be fully evaluated.
The expression of selected miRNAs is profoundly
altered in many cancers, including HCC, suggesting their
potential importance as biomarkers and therapeutic tar‑
gets (FIG. 5). In addition to patient-to‑patient and disease
variability, these analyses use different approaches and
starting materials for analyses (HCC versus non-tumour
liver or HCC compared with uninfected liver). Although
the mechanisms by which HBV and HCV alter miRNA
expression remain to be elucidated, many miRNA genes
are located at fragile sites or in cancer-associated genomic
regions, suggesting that the genetic instability that is
associated with these chronic infections may alter the
expression of these miRNAs148. The potential of miRNAs
as biomarkers derives from an appreciation that most
miRNAs target many genes, and that as cancer is a
multi-step process, altered expression of crucial miRNAs
will probably have diagnostic or prognostic value.
Selected miRNAs may also be therapeutic targets, such as
miR‑122, which stimulates HCV replication. Currently,
a miR‑122 antagonist (Miravirsen (SPC3649; Santaris
Pharma A/S)) is being evaluated in patients with chronic
HCV, and preliminary results have shown considerable
and prolonged anti-viral activity (see the ClinicalTrials.
gov website; see Further information). This approach
holds promise for the treatment of HCV, and it is hoped
that crucial pathways in tumorigenesis could be similarly
targeted. However, as miR‑122 can be a tumour suppres‑
sor, blocking its expression may increase the risk of HCC
in patients who are chronically infected149,150.
In serum, elevated AFP >400 ng per ml has been
used as an early marker of HCC151, although it lacks
sensitivity and is not specific for virus-associated HCC.
NATURE REVIEWS | CANCER VOLUME 13 | FEBRUARY 2013 | 131
© 2013 Macmillan Publishers Limited. All rights reserved
REVIEWS
Des-γ-carboxy prothrombin (DCP) and Lens culi‑
naris agglutinin-reactive fraction of AFP (AFP‑L3)
have a higher specificity and a better negative pre‑
dictive value compared with total AFP. This combi‑
nation has identified patients with negative imaging
results for tumours who would benefit from follow‑up
evaluation152. Recently, glypican‑3 (GPC3), which is a
membrane-bound heparin sulphate proteoglycan, was
found to be expressed in HCC cells and tissues, and to
some extent in hepatocytes. Although GPC3 seems
to be an early marker of HCC153, it is not clear whether
it is virus associated. Serum GPC3 levels were
increased in most patients with early-stage HCC, and
an assay combining GPC3 with AFP has increased
sensitivity for the detection of HCC at all stages. Other
potential serum biomarkers for HCC include heat
shock protein 27 (HSP27), α2‑HS glycoprotein, apoli‑
poprotein E and apolipoprotein M, and several autoan‑
tibodies154.Unfortunately, most of these have not been
validated in larger studies, and given that HCC is
heterogeneous in terms of markers, it is not clear
which marker or markers are going to be useful for
early detection.
Additional attempts to identify biomarkers have used
microarrays of tumour compared with non-tumour or
uninfected liver and cirrhotic liver compared with livers
with dysplastic and/or regenerative nodules. Few con‑
sistent differences have been found155–160, and these
differences have not been validated by larger studies.
Differences might also be due to the techniques used,
prior treatment, stage of tumour differentiation, encap‑
sulation, vascularization, extent of fibrosis or inflamma‑
tion and whether the analysed samples were from primary
tumours or intrahepatic metastases. Further, it is not clear
whether these studies identify markers of early HCC or
of tumour progression. Biomarkers have also been sug‑
gested from studies on cell lines and from animal mod‑
els of HBV and HCV, but there has been little large-scale
validation using human clinical samples. However, recent
work has identified a single nucleotide polymorphism
(SNP) in EGF that is associated with an increased risk of
HCC among patients with HCV161,162. An SNP in inter‑
leukin‑28B (IL28B) also influences the outcome of HCV
infection163 and the response of patients to treatment 164.
By contrast, it is not clear that SNPs in EGF and IL28B
have any role in the pathogenesis of HBV infection165–167.
By comparison, SNPs in TGFB1 may increase the risk of
HCC in both infections168,169. Thus, there is a potential role
for SNPs as biomarkers.
HCC is not detected early enough for curative inter‑
vention. Available therapies are not tumour specific
and are not very effective when HCCs are diagnosed
at a late stage. The first drug found to increase sur‑
vival, sorafenib, does so by inhibiting cell proliferation
(via the RAS–RAF–ERK pathway) and by blocking
angiogenesis (via the VEGFR and platelet-derived
growth factor receptor). HCC is also associated with
the activation of EGFR, IGFR, WNT–β‑catenin and the
PI3K–AKT–mTOR pathways 51,53,170–175, which may
underlie sorafenib resistance, suggesting that combined
therapies will be required to have a sustained effect.
REVIEWS

There are more than 50 drugs in almost 200 clinical
trials that are currently underway or that have recently
been completed in patients with HCC176 but it is not
yet clear what pathways support HCC growth. In this
context, it will be important to target pathways that are
involved in oncogene addiction, as these drive tumour
growth. HBV-associated HCC seems to be addicted
to the RAS–ERK and PI3K–AKT–mTOR pathways87.
However, it is not clear what pathways define onco‑
gene addiction in HCV-mediated HCC. The transcrip‑
tion factor LSF, which promotes cell cycle progression
through G1/S phase, may be part of another onco‑
gene addiction pathway in HCC177. WNT–β‑catenin
and ERBB2 signalling are constitutively activated in
HCC79,87, but it is not clear whether HCC is addicted
to these pathways. Given the heterogeneity of HCC,
treatment cocktails will need to be developed for dif‑
ferent subsets of patients depending on their bio‑
marker profiles. As clinical trials cannot be conducted
over many years or decades, biomarkers may be used
as surrogate end points in evaluating drug efficacy.
This should accelerate the evaluation of new drugs in
patients with CLD who are at a high risk of HCC before
tumour appearance, and may allow the development of
preventive chemotherapeutics.
Conclusions
HBV and HCV trigger changes in gene expression in
the liver that confer the hallmarks of cancer on cells.
These changes result from alterations in DNA methy­
lation, changes in miRNA expression profiles and the
constitutive activation of numerous signal transduc‑
tion pathways. The induction of EMT in cirrhotic or
dysplastic and early HCC nodules is associated with a
hypoxic environment that favours the survival and out‑
growth of cells with stem cell-like characteristics that
promote tumour development and progression. The
pathogenesis of HCC is immune mediated, and the
oxidative environment created in the liver by immune
responses is exploited by these viruses to constitutively
activate signalling pathways that block apoptosis, pro‑
mote survival, support HBV and HCV replication,
and promote virus persistence. These viruses also
delay or block innate immunity, thereby compromis‑
ing the development of adaptive immune responses.
This provides opportunities for the development of spe‑
cific immunotherapeutics and/or therapeutic vaccines
that would help to resolve virus infection and block
CLD progression. Immunotherapy may also be com‑
bined with nucleoside analogues and other inhibitors
of virus replication in the control of these infections.
A cure may be possible for HCV because it exists solely
as an episome, but for HBV, proteins made from inte‑
grated virus templates may still trigger and promote
CLD even if episomal DNA (which includes linear
replicative forms and covalently closed circular DNA
(cccDNA)) is cleared. The identification of signalling
pathways and gene targets that are common to the
pathogenesis of HBV and HCV infections may provide
opportunities for the application of existing drugs and
the development of new drugs that will be effective
against both infections. Further, it is likely that simulta‑
neously targeting multiple pathways that drive tumour
progression will provide opportunities to develop spe‑
cific therapies. The importance of developing treat‑
ments for patients who are chronically infected before
tumour appearance is underscored by the reality that
most of the changes in the liver before tumour develop‑
ment are reversible by drugs, but many of the changes
in tumour nodules involve mutations in pathways that
are much more difficult to treat.
Importantly, virus proteins trigger changes in sig‑
nalling pathways and gene expression in hepatocytes
that are often found to be mutated in HCC178. In such
a model, which has been proposed for other tumour
types, epigenetic modification of tumour suppressor
genes may permit the constitutive expression of onco‑
genes in early tumorigenesis, and mutation in these
same oncogenes results in higher constitutive expres‑
sion that is characteristic of tumour progression179.
Thus, oncogene addiction may be an early event in
cancer. Inflammation and ROS would promote this
addiction. As cell clones expand, they acquire her‑
itable epigenetic changes that result in a permanent
change in phenotype179, and take on increasing fea‑
tures of tumour cells. These changes will be fertile
ground for the establishment of biomarkers and
targets for therapeutic intervention. Fortunately,
drugs are already being developed to many of these
epigenetic targets180.

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Acknowledgements
This work was supported by US National Institutes of Health
(NIH) grants AI076535 and CA104025 to M.A.F.
Competing interests statement
The authors declare no competing financial interests.
FURTHER INFORMATION
Mark A. Feitelson’s homepage: http://www.temple.edu/cst/
research/profiles/biology/feitelson.html
ClinicalTrials.gov: http://clinicaltrials.gov/
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